Summary: In addition to the high affinity IgE receptor (Fc?RI), human connective tissue mast cells (MCTC) express a recently described G protein coupled receptor (GPCR), known as Mas-related GPCR-X2 (MRGPRX2). Our lab was the first to demonstrate that host defense antimicrobial peptides (HDPs) activate human mast cells via MRGPRX2, which likely contributes to innate immunity. Emerging evidence suggests that MRGPRX2- mediated local mast cell activation clears bacterial infection via the recruitment of neutrophils and promotes adaptive immunity to control reinfection. However, dysregulation of host defense and excessive generation of the host defense peptide, LL-37 contributes to the pathogenesis of rosacea. MrgprB2 is the mouse counterpart of human MRGPRX2 and our preliminary data demonstrated that LL-37-mediated experimental rosacea is significantly reduced in MrgprB2-/- mice when compared to wild-type mice. MRGPRX2 also serves as a novel GPCR for neuropeptides such as substance P, hemokinin-1 and pituitary adenylate cyclase- activating peptide (PACAP). Furthermore, MRGPRX2 is an ?atypical opioid receptor? and some of the side effects opioids are likely mediated via mast cell degranulation through this receptor. However, the molecular mechanisms involved in the activation and regulation of its downstream signaling remains largely unknown. In addition to G proteins, many GPCR agonists also signal via the recruitment of adapter proteins known as ?-arrestins. This pathway not only contributes to GPCR desensitization but also provides a platform for a variety of G protein-independent signaling. Biased GPCR agonists preferentially activate pathways mediated by G proteins (G protein-biased) or ?-arrestins (?-arrestin-biased). Agonists that activate both pathways are known as balanced agonists. Recently, we made the surprising observation that while compound 48/80 activates both G protein and ?-arrestin (balanced agonist) an angiogenic host defense peptide activates only G protein but not ?-arrestin (G protein-biased agonist). Based on these findings, we hypothesize that balanced and G protein-biased MRGPRX2 agonists activate mast cells via different mechanisms to promote distinct biological responses in vivo. Because MrgprB2 is the mouse counterpart of the human MRGPRX2, we will incorporate this receptor for many of our in vivo studies. In aim #1, we will determine which of the known mast cell secretagogues (HDPs, neuropeptides, opioids and FDA-approved pseudo-allergic drugs) act as balanced and G protein-biased agonists for MRGPRX2. We will also identify the structural determinants on the receptor that facilitate their coupling to G protein and ?-arrestins. In aim #2, we will modulate host defense, pseudo-allergy and rosacea by targeting MRGPRX2/MrgprB2?s balanced/G protein biased signaling in mast cells in vivo. In aim #3, we will determine the roles of ?-arrestin1 and ?-arrestin2 on MRGPRX2 and MrgprB2-mediated signaling in vitro and biological responses in vivo. Completion of this study will provide novel approaches to modulate MRGPRX2-mediated host defense and inflammatory diseases.